Abstract

The immunoresponsive gene 1 (IRG1) protein has crucial functions in embryonic implantation and neurodegeneration. IRG1 promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species (ROS). The cytoprotective protein heme oxygenase-1 (HO-1), which generates endogenous carbon monoxide (CO), is expressed in the lung during Lipopolysaccharide (LPS) tolerance and cross tolerance. However, the detailed molecular mechanisms and functional links between IRG1 and HO-1 in the innate immune system remain unknown. In the present study, we found that the CO releasing molecule-2 (CORM-2) and chemical inducers of HO-1 increased IRG1 expression in a time- and dose-dependent fashion in RAW264.7 cells. Furthermore, inhibition of HO-1 activity by zinc protoporphyrin IX (ZnPP) and HO-1 siRNA significantly reduced expression of IRG1 under these conditions. In addition, treatment with CO and HO-1 induction significantly increased A20 expression, which was reversed by ZnPP and HO-1 siRNA. LPS-stimulated TNF-α was significantly decreased, whereas IRG1 and A20 were increased by CORM-2 application and HO-1 induction, which in turn were abrogated by ZnPP. Interestingly, siRNA against IRG1 and A20 reversed the effects of CO and HO-1 on LPS-stimulated TNF-α production. Additionally, CO and HO-1 inducers significantly increased IRG1 and A20 expression and downregulated TNF-α production in a LPS-stimulated sepsis mice model. Furthermore, the effects of CO and HO-1 on TNF-α production were significantly reversed when ZnPP was administered. In conclusion, CO and HO-1 induction regulates IRG1 and A20 expression, leading to inhibition of inflammation in vitro and in an in vivo mice model.

CORM-2 and CoPP/hemin-induced expression of IRG1 is dependent on HO-1 in RAW264.7 macrophages. (a) RAW264.7 cells were treated with CORM-2 (0, 5, 10 and 20 µM) or its negative control, RuCl3 (10 and 20 µM) for 8 h and the levels of IRG1 and HO-1 mRNA were detected by using RT-PCR analysis. (b–d) Cells were pre-treated with ZnPP (10 and 20 µM) for 0.5 h and treated with CORM-2 for 8 or 16 h. The levels of IRG1 (b) and HO-1 (c) mRNA were measured at 8 h by real-time RT-PCR and (d) IRG1 protein levels were determined at 16 h by western blot analysis. (e) Cells were transfected with HO-1 siRNA or control siRNA (Con siRNA). After treatment with 20 µM CORM-2 for 16 h, cells were harvested and protein levels of HO-1 and IRG1 were performed by western blotting. (f and g) Cells were pre-treated with ZnPP (20 µM) for 0.5 h and treated with CoPP (10 µM) and hemin (10 µM) for 8 or 16 h. (f) IRG1 mRNA was measured at 8 h by real-time RT-PCR and (g) IRG1 protein levels were determined at 16 h by western blot analysis. The representative bands or blots are shown. Data represent mean±s.e.m., * P<0.05 and ** P<0.001 as compared with control and ##P<0.001 as compared with the cells exposed to only CORM-2, hemin or CoPP.

CORM-2 and hemin increases A20 expression via HO-1 activation in RAW264.7 macrophages. (a) RAW264.7 cells were incubated with CORM-2 (20 µM) for 0, 2, 4, 8, 16 and 24 h, and western blot analysis was performed to detect A20 expression. (b and c) Cells were treated with CORM-2 (0, 5, 10, 20 and 40 µM) for 8 and 24 h and then, the levels of (b) A20 protein and (c) A20 mRNA were measured by western blot analysis and real-time RT-PCR analysis, respectively. (d and e) Cells were pre-treated with ZnPP (20 µM) for 0.5 h and further incubated with CORM-2 (10 and 20 µM) for 8 or 24 h. (d) The levels of A20 mRNA were analyzed at 8 h by real-time RT-PCR. (e) A20 protein level was carried out at 24 h by western blot analysis. (f) Cells were transfected with HO-1 siRNA or control siRNA (Con siRNA). Cells were treated with 20 µM CORM-2 for 24 h, and cells were harvested and protein levels of HO-1 and A20 were performed by western blotting. (g) Cells were treated with hemin at various doses for 8 h, and the levels of A20 mRNA were analyzed by real-time RT-PCR. (h) Cells were pre-treated with ZnPP (20 µM) for 0.5 h and hemin (10 and 20 µM) was treated for 24 h, and then A20 protein level was analyzed by western blot analysis. The representative bands or blots are shown. Data represent mean±s.e.m., ** P<0.001 as compared with control; and ##P<0.001 as compared with the cells exposed only to CORM-2, respectively.

CORM-2/hemin-induced IRG1 decreases inflammation via A20 expression in RAW264.7 macrophages. (a and b) RAW264.7 cells were pre-treated with ZnPP (20 µM) for 0.5 h and then administrated with CORM-2 (20 µM) for 1 h and further incubated with LPS (100 ng/ml) for 24 h. (a) Cell supernatants were analyzed by ELISA to measure TNF-α. (b) Cell lysates were subjected western blot analysis for protein level of IRG1 and A20. (c) Cells were transiently transfected with IRG1 siRNA or control siRNA (Con siRNA). After treatment with CORM-2 (20 µM) for 1 h, the cells were stimulated with LPS (100 ng/ml) for 24 h, and the levels of TNF-α, A20 and IRG1 mRNA were analyzed by real-time RT-PCR. (d) Cells were transfected with A20 siRNA or control siRNA (Con siRNA). Following treatment with 20 µM CORM-2 for 1 h, the cells were stimulated with 100 ng/mL LPS for 24 h, the levels of TNF-α and A20 mRNA were analyzed by real-time RT-PCR. (e and f) Cells were pre-treated with ZnPP (20 µM) for 0.5 h and then administrated with hemin (10 µM) for 1 h and incubated with LPS (100 ng/mL) for 24 h. (e) Cell supernatants were subjected for ELISA to measure TNF-α. (f) Cell lysates were subjected to western blot analysis to determine the protein level of IRG1 and A20. (g) Cells were transfected with IRG1 siRNA or control siRNA (Con siRNA). After treatment with 10 µM hemin for 1 h, the cells were stimulated with 100 ng/ml LPS for 24 h, and the levels of TNF-α, A20 and IRG1 mRNA were analyzed by real-time RT-PCR. (h) Cells were transfected with A20 siRNA or control siRNA (Con siRNA). After treatment with 10 µM hemin for 1 h, the cells were stimulated with 100 ng/ml LPS for 24 h, and the levels of TNF-α and A20 mRNA were analyzed by real-time RT-PCR. Data represent mean±s.e.m., * P<0.05 and ** P<0.001 as compared with control; and #P<0.05, ##P<0.001 as compared with the cells exposed to only LPS (normal condition or IRG1/A20 siRNA, compared separately) and ns, non-significant as compared with only LPS (IRG siRNA or A20 siRNA).

Inhalation of CO gas and administration of CORM-2 and hemin increase IRG1 and A20 expression via HO-1 expression and decrease inflammation in a sepsis mouse model in vivo. (a–c) Wild-type 7-week-old male C57BL/6 mice were inhaled with CO gas (250 ppm) for 6 days (4 h daily basis) and some of them were administrated with ZnPP (5 mg/kg; i.p.). After 6 days, the mice were administrated with LPS (12.5 mg/kg; i.p.) for 16 h. (a) Blood serum was analyzed for the TNF-α level by ELISA. (b) Liver tissues were analyzed for the levels of TNF-α by real-time RT-PCR. (c) Liver tissues were analyzed for the levels of IRG1, A20 and HO-1 mRNA by real-time RT-PCR. (d–f) Wild-type 7-week-old male C57BL/6 mice were pre-treated with CORM-2 (30 mg/kg; i.p.), hemin (10 mg/kg; i.p.) and ZnPP (5 mg/kg; i.p.) for 2 h and, then, the mice were administrated with LPS (12.5 mg/kg; i.p.) for 16 h. (d) Blood serum was analyzed for the TNF-α level by ELISA. (e) Liver tissues were analyzed for the levels of TNF-α by real-time RT-PCR. (f) Liver tissues were analyzed for the levels of IRG1, A20 and HO-1 mRNA by real-time RT-PCR. Data represents mean±s.e.m., * P<0.05 and ** P<0.001 as compared with control; and #P<0.05 and ##P<0.001 as compared with the cells exposed to LPS+CO/CORM-2/hemin (in case TNF-α, ##P<0.001 as compared with the cells exposed to only LPS).